Biological compatibility of scion and rootstock in pomiculture

The success of the union and subsequent growth of a graft depends on the degree of compatibility between the scion and the rootstock. In literature, when characterizing biological compatibility, terms such as physiological compatibility, biological correspondence, affinity, affinity, sympathy, and congeniality are used. However, we consider the term "biological (physiological) compatibility of components" the most appropriate for denoting this complex phenomenon. The most precise definition of "compatibility" and "incompatibility" of fruit crop grafting components was given in the work of G. V. Trusevich (1964). According to his interpretation, "compatibility of the rootstock and scion... refers to their ability to form and maintain, over a long period, an anatomically correct and mechanically strong union that ensures successful metabolism between them and the normal course of the grafted plant's life processes (growth and fruiting)." The term "incompatibility" combines a number of cases where the compatibility of grafting components is unsatisfactory from the point of view of production requirements for grafting combinations, or is completely absent.

Since there are significant losses associated with the death of scion-rootstock combinations in the nursery and the orchard due to incompatibility, many researchers have attempted to establish the nature of this phenomenon and thereby learn to predict it in the first year of the grafted plants' life, before planting them in a permanent location.

Possible causes of incompatibility in the grafting of fruit plants and methods of overcoming it

In pomology, it was long believed that one of the reasons for incompatibility could be the genetic distance between components. As a rule, grafts of plants belonging to different species, genera, and families are less successful.

While not denying a certain relationship between the degree of botanical affinity and the compatibility of grafting components, it should be recognized as very approximate. For example, there is insufficient compatibility of some apple cultivars with seedlings of various forms of Chinese crabapple, Caucasian apple, Babaarab apple, etc. At the same time, interspecific and even intergeneric grafts are quite viable and widely used in production: Lyubskaya cherry on sweet cherry, some pear cultivars on quince (Korovin, 1979; Tatarinov, Zuev, 1984).

As can be seen from the listed facts, botanical proximity cannot serve as a reliable criterion for the compatibility of grafting components. According to D. M. Grodzinsky et al. (1984), this is due to the fact that botanical classification is based on reproductive traits, whereas grafting relates primarily to the vegetative functions of plants.

According to the classification proposed by G. V. Trusevich (1964), four forms of incompatibility of grafting components are observed in fruit crops:

1) complete inability of the scion and rootstock to unite during grafting (complete incompatibility);

2) formation of a short-lived connection between the scion and rootstock using callus masses (callus bridges) without the connection of conductive elements;

3) incomplete or temporary connection of the cambium and conductive elements of the scion and rootstock; the grafted plants initially grow normally, but later cases of breakage at the union site or premature death are possible;

4) the union of the rootstock and scion is anatomically correct and strong, no breakages are observed, but the plants are characterized immediately or subsequently by weakened growth, low productivity, and a short lifespan (a phenomenon noted when grafting some sweet cherry cultivars onto St. Lucie cherry).

According to G. V. Trusevich (1964), the incompatibility of grafting components in the third and fourth cases can be considered delayed.

In grafted apple plants, V. A. Korovin (1979) discovered three forms of manifestation of scion-rootstock incompatibility:

1) weak union of the scion and rootstock;

2) spot disease of the rootstock;

It has been established, however, that the incompatibility of grafting components, in whatever form it appears, is caused by reasons of a biological (physiological) nature. Therefore, in any case, one should speak of physiological incompatibility as a broad concept that includes several forms of its manifestation. The latter, in all likelihood, may be caused by the action of different mechanisms.

Judging by the literature, the phenomenon of tissue incompatibility between scion and rootstock is studied to a greater extent in fruit crops. This covers the first to third forms of incompatibility manifestation according to the classification of G. V. Trusevich (1964) and the first form according to the classification of V. A. Korovin (1979). Randi indicates several common symptoms of tissue incompatibility of grafting components in woody plants: absence of callus cell formation; formation of callus, but subsequent incomplete differentiation of conductive tissues; necrosis of cells on the cut surface at the contact point of the scion and rootstock. In the union zone of incompatible grafting components, a significant amount of loose parenchyma cells is deposited, an abscission layer is formed (Grodzinsky, 1984; 1985), and active rejection of scion tissues occurs. Such a picture is observed, for example, in the grafting combination of the Antonovka Obyknovennaya apple cultivar on Chinese crabapple, as well as when grafting some pear cultivars onto quince, peach onto apricot and plum, sweet cherry onto bird cherry, etc.

In case of incompatibility of the delayed type, fractures of grafted plants are observed in the orchard, with the emergence of foci of unorganized growth and the formation of a layer in the boundary area of the grafting components. According to I.F. Gavryushova (1984), the trigger for such growth is the mismatch between the scion and rootstock in the rhythms of nutrient accumulation and consumption, as well as enzyme activity.

Defects in the contact zone of incompatible grafting components are the cause of impaired movement of water and nutrients through the union site and their uneven distribution in the scion and rootstock (Tatarinov, 1976; Sîrbu, Shishkanu, 1982; Sîrbu, Stoyanov, 1984).

In recent years, significant progress has been made in understanding the mechanisms that determine tissue incompatibility.

A hypothesis has been proposed regarding the identity of the processes occurring during transplants and the introduction of parasites into a plant. According to researchers, in both the first and second cases, mechanisms of recognizing "self" from "non-self" operate to reject anything foreign. In other words, the basis of tissue incompatibility of grafting components lies in the ability of somatic cells to engage in mutual (protein-protein) "recognition" (Grodzinsky, 1984, 1985; Waring, Phillips, 1984).

Evidence for this is provided by experimental results indicating the possibility of overcoming tissue incompatibility by suppressing the universal plant defense mechanism through the action of ionizing radiation or protein synthesis inhibitors. This confirms the immunobiological nature of this manifestation of rootstock-scion incompatibility.

In the process of biological "recognition" of cells, a special role is played by lectins, which are a group of proteins characterized by the general property of selective binding of sugars or sugar-containing macromolecules, in particular glycoproteins (Lutsik et al., 1981; Markov, Khavkin, 1988; Waring, Phillips, 1984; Bowles, Kauss, 1975; Goldstein, Hayes, 1978).

Based on the data presented, a conclusion has been drawn about the potential of using immunochemical methods for predicting tissue incompatibility. However, the labor-intensive nature of such methods will likely limit their use for mass evaluation of samples.

Another fairly common form of physiological incompatibility of grafting components, most frequently encountered in apple trees, is rootstock pitting (Korovin, 1979; Tatarinov, Zuev, 1984).

Plants with rootstock pitting are, as a rule, stunted while still in the nursery (they have weak shoots and small, dryish leaves) and are subject to culling.

However, there are known cases where this form of scion-rootstock incompatibility is the cause of mass tree mortality in the orchard. Its characteristic feature is the formation of necroses in the bark and wood of the rootstock. At the same time, no disturbances are observed in the scion tissues.

With severe incompatibility of grafting components, necroses are also noted at the union site, and with its mild manifestation, uneven wood deposition or "wood rippling" can be detected.

Consequently, identifying this form of mismatch between grafting components requires carrying out quite labor-intensive morphological studies using histological methods.

It should be noted that the opinions found in the literature regarding the causes of rootstock pitting are highly contradictory. For instance, G.V. Trusevich (1964) views it as a specific functional disease caused by the fungus *Fusarium* infecting the rootstock.

At the same time, V.A. Korovin (1979) provides a series of facts proving that this fungus has no relation to the emergence of rootstock pitting. The latter, in his opinion, is caused by the influx of certain toxic substances from the scion, which trigger the dieback of cambium, phloem, and rootstock wood tissues. It seems likely that one can agree with this conclusion, especially since a highly toxic agent—prunasin—has already been detected at the contact site of some incompatible components.

According to G.V. Trusevich (1964), rootstock pitting has also been recorded in pear plants.

Low fat and starch content in the rootstock combined with intense polysaccharide accumulation in the scion is a hallmark of physiological incompatibility of apple grafting components of the rootstock starvation type. A similar phenomenon is observed when grafting various pear cultivars from the middle latitudes onto quince rootstocks, as well as some peach cultivars onto the Damas-1869 plum rootstock.

In nursery plants and trees with this form of physiological incompatibility of grafting components, the following changes are noted:

• dieback of rootlets by the end of summer;
• early mass formation of fruit buds.

It is assumed that the low starch content in the rootstock may be caused not only by the impaired outflow of sugars from the scion to the rootstock due to defects at the contact site, but also by the disruption of polysaccharide synthesis, even when such defects are absent and sugars enter the rootstock in significant quantities.

It should, however, be noted that despite the specific features of one or another form of physiological scion-rootstock incompatibility, in practice it is sometimes difficult to draw a clear line between them. There are known cases, for example, where, with a severe degree of incompatibility of grafting components of the rootstock pitting or starvation type, defects in the union zone typical of tissue incompatibility are observed.

To identify the incompatibility of a scion and a rootstock, visual methods are still widely used in nursery practice. Discrepancies between grafting components are judged by a number of signs:

• early cessation of growth of the nursery plants;
• premature change in leaf color;
• leaf fall (Chang, 1939; Trusevich, 1964; Nesterov, 1968; Kolesnikova et al., 1984; Indenko, 1987, etc.).

However, the listed signs can also manifest under the influence of certain extreme factors on plants, which makes their use for predicting incompatibility difficult. Thickening of one of the components at the grafting site is often associated with a discrepancy between the scion and the rootstock. At the same time, there is a diametrically opposite opinion. In particular, S. N. Stepanov (1956) concludes that a greater thickness of the rootstock compared to the scion is a completely normal phenomenon, indicating good nutrition of the grafting components. Apparently, the indicated indicator also cannot be used to predict the discrepancy of grafting components.

For a quick assessment of the compatibility of rootstocks with various cultivars in nursery conditions or for breeding purposes, V. A. Korovin (1979) proposed a method of transplanting rootstock bark rings onto annual or perennial branches of apple cultivars, which allows identifying the compatibility of grafted components 2.5–3.0 months after the specified grafting.

We believe that only physiological and biochemical parameters that most accurately characterize the functional state of cultivar-rootstock combinations and correlate with their compatibility (incompatibility) should be used as diagnostic criteria. This should be kept in mind first and foremost when identifying delayed-type incompatibility, which is impossible to establish by external signs in young plants.

With any form of discrepancy between grafting components, disturbances in the coordination and synchrony of metabolic processes are noted in them, indicating that the organism does not function as a single unit.

It is no coincidence that one of the methods for determining the degree of compatibility of grafting combinations involves comparing the daily growth rhythms of shoots of the grafted cultivar and those from the dormant bud of the rootstock (Troysky, Abolin, 1962). Different adaptive reactions of incompatible scion and rootstock under the influence of adverse factors are also indicated (Grinenko, Butner, 1965; Veselova et al., 1973; Pudrikova, Dorokhov, 1984; Grinenko et al., 1985).

However, far from all characteristics are suitable for judging the similarity or difference in the intensity and direction of individual metabolic processes in grafting components. In particular, the determination of catalase activity in the scion and rootstock, as proposed by I. V. Kaimakan (1958), does not always lead to the achievement of the set goal.

Obviously, the problem of early diagnosis of the biological correspondence of grafting components requires a correct methodological approach to its solution. V. I. Safonov and A. E. Weidenberg (1969) point to the possibility of predicting the compatibility of cultivars and rootstocks by the degree of identity of bark protein systems, expressed by a "kinship coefficient". Meanwhile, the specified method does not provide for grafting. Such a solution to the issue is hardly legitimate, because in this case, the result of the mutual influence of grafting components occurring in the newly created plant organism is not taken into account.

This drawback is eliminated if diagnostic parameters of the tested cultivar-rootstock combination are compared with those of grafted plants with a known degree of scion and rootstock compatibility. As a control, it is advisable to use:

• a highly compatible grafting combination obtained as a result of homotransplantation;
• a variant with "self" grafting.

The assessment of the compatibility of grafting components is no less accurate when comparing various physiological and biochemical parameters of a cultivar-rootstock combination with the same parameters of the corresponding non-grafted rootstock (Korovin, 1979; Titova, 1984; Piskorskaya, Titova, 1986). Moreover, V. A. Gryazev (1981) recommends using the method of separately studying the properties of grafting components in comparison with their various combinations when considering all issues of the physiology of a grafted plant.

In all likelihood, only such a solution to the set task will allow taking into account the nature of the changes occurring in the scion or rootstock as a result of grafting, and, consequently, the prospects of the scion-rootstock combination. Using the listed methodological approaches to studying the phenomenon of incompatibility of grafting components, researchers have attempted to determine physiological and biochemical parameters non-specifically associated with its various forms of manifestation.

The possibility of early diagnosis of discrepancy between scion and rootstock has been established, taking into account the peculiarities of the accumulation of the following indicators in plant organs and tissues:

• carbohydrates (Trofanyuk, 1971; Syrbu, Ioltukhovsky, 1973; Titova, 1984; Titova, Khachaturyan, 1984);
• total and protein nitrogen;
• mineral nutrients (Syrbu, Stoyanov, 1984);
• free amino acid content (Tsurkan et al., 1978; Syrbu et al., 1979; Piskorskaya, Titova, 1986);
• phosphorus compounds;
• activity of catalase enzymes (Subbotina, Andryushchenko, 1969; Korovin, 1979), peroxidase and polyphenol oxidase (Titova, Shishkanu, 1984).

To evaluate the degree of grafting component compatibility, it is recommended to use indicators of water exchange and photosynthetic activity of plants (Veselova et al., 1973; Korovin, 1979; Suvak, Shishkanu, 1984). Attempts have been made to predict the incompatibility of the scion and rootstock based on isoperoxidase spectra in leaves and bark. Another method for determining the physiological compatibility of grafting components in fruit crops involves comparing the protein spectrum in the roots of a cultivar-rootstock combination with that of the corresponding non-grafted rootstock.

Scion-rootstock incompatibility often manifests not immediately, but several years after planting the orchard. Such delayed incompatibility causes enormous damage to nurseries and horticultural enterprises, as it leads to the death of already mature trees. Traditional diagnostic methods based on individual metabolic indicators do not provide a reliable forecast. To identify the problem in time, a new rapid method for assessing compatibility based on the RNA/DNA ratio in growth points has been developed.

Biological principle of compatibility assessment

The diagnostic is based on a simple biological pattern. In a compatible cultivar-rootstock combination, metabolic activity is at the level of a healthy non-grafted rootstock. In the event of a physiological conflict, the coordination of functions is disrupted, and the metabolic activity of the grafted plant begins to differ from the initial rootstock. The stronger the incompatibility, the greater the difference in the metabolic activity of the graft and the pure rootstock.

For an accurate assessment, the compatibility coefficient (K) is calculated based on the difference in the RNA/DNA ratios in the combination and the rootstock: K = (RNA/DNA)_combination – (RNA/DNA)_rootstock. If the coefficient value exceeds a critical threshold, the plants from the nursery are culled. In practice, the boundary between average and poor compatibility is often blurred, so both groups are subject to culling.

Procedure for sampling and analysis

To carry out the diagnostics, experimental grafts are planted, leaving a portion of the rootstocks non-grafted as a control. In the nursery, shoot tips are sampled in the second field during the active growth phase. In the conditions of the North Caucasus, this period falls at the end of May — beginning of June. To speed up the assessment, winter grafting under controlled conditions can be used: in this case, analyses start 1.0–1.5 months after the beginning of the growing season.

1. Separate shoot tips 4.0–10.0 mm in length with apices (growth points). For analysis, take samples from 10–15 plants of average development for each combination and the control non-grafted rootstock.
2. From each pooled sample, weigh out 0.05–0.10 g aliquots.
3. Transfer each aliquot into a test tube containing 5 ml of 96% ethyl alcohol.
4. Boil the tubes with samples for 5 minutes to fix the material.
5. Perform chemical separation of RNA and DNA using the classical method, then determine their concentration in the solution spectrophotometrically.

When conducting laboratory analyses, strictly observe the number of repetitions. For vegetatively propagated rootstocks, two-fold analysis repetition is required. For seed rootstocks, due to their genetic diversity, the repetition must be increased to four-fold.

• Shoot tip length — 4.0–10.0 mm
• Aliquot mass — 0.05–0.10 g
• Volume of 96% ethyl alcohol — 5 ml
• Boiling time for fixation — 5 min
• Critical value of coefficient K — 0.18

Thus, the proposed method is universal. Along with this, it is highly reliable. The validity of the assessment results for the compatibility of grafting components is ensured by the choice of the most informative diagnostic indicator, which is determined in the regulatory center of the plant. As a result, the metabolic activity of the plant organism as a whole is taken into account. Furthermore, when diagnosing the compatibility of a cultivar-rootstock combination using the proposed method, the fact of the influence of one grafting component on the other is considered. The most convincing proof of the reliability and accuracy of the discussed method is the complete coincidence of the conclusions regarding the degree of compatibility of cultivar-rootstock combinations made based on the results of early diagnosis (at the beginning of the assessment, the degree of compatibility of the tested rootstocks with regionalized cultivars was still unknown) and the results of agrobiological observations of the state of grafted plants several years after transplanting from the nursery to a permanent site. Some examples of such comparison are given in table 2.

For plants obtained through double grafting, it seems appropriate to assess the biological compatibility of the grafting components in two stages:

• In the first stage, the compatibility of the intermediate insert with the main rootstock is predicted.
• In the second stage, the compatibility of the cultivar with the intermediate insert is predicted.

In the first case, a grafted plant consisting of two components (an intermediate rootstock/main rootstock combination) is tested. In the second case, a grafting combination represented by three components (scion, intermediate rootstock, and main rootstock) is considered, and the combination of the intermediate rootstock/main rootstock is conventionally accepted as the rootstock.

The results of our research indicate that apple tree intercalary rootstocks M9, M27, and SK3 are highly compatible with the root-forming rootstock – seedlings of the Anis Kubansky cultivar. The same can be said about the compatibility of apple cultivars Renet Simirenko, Idared, Korey and the intercalaries (K < 0.18).

Incompatibility between scion and rootstock in the nursery, and subsequently in the orchard, is primarily prevented by the correct selection of grafting components. Having a large set of cultivars and rootstocks, it is necessary to choose the most viable combinations from them, thereby eliminating any possibility of component mismatch. To this end, it is advisable to use the aforementioned methods of early (in the first year of life) compatibility diagnostics of cultivar-rootstock combinations.

Thorough culling of plants with even the slightest signs of scion-rootstock incompatibility is mandatory. All plants exhibiting the following signs are subject to destruction:

• premature discoloration of leaves;
• rosetting and small-leaf symptoms;
• affected by pitting disease of the rootstock, accompanied by growth retardation.

In plants with this form of component incompatibility, streaks or spots of black, brown, or gray color are visible on longitudinal root sections.

It is practically impossible to eliminate incompatibility in orchard trees. As an exception, bridge grafting is recommended for pear trees grafted onto quince that show signs of grafting component incompatibility. However, this technique is quite labor-intensive.

Complete removal of the scion and grafting of a compatible cultivar onto a rootstock weakened by starvation usually yields very mediocre results. The incompatibility of many pear cultivars with quince is overcome by grafting an intermediate insert between them – a cultivar that performs well on quince. The use of an intermediate insert may prove useful for apple trees and other fruit crops (Tatarinov, Zuev, 1984).

• What is biological compatibility?
• List the forms of manifestation of grafting component incompatibility in fruit crops according to the classifications proposed by G. V. Trusevich and V. A. Korovin.
• Indicate the possible causes of incompatibility in fruit plant grafting.
• List the diagnostic criteria for scion and rootstock incompatibility.
• What ways of overcoming grafting component incompatibility do you know?